This proposal involves an investigation of two homologous polypeptide protease inhibitors (Mr ca. 5,000) from potatoes. One of these inhibits the pancreatic carboxypeptidases and the other affects chymotrypsin. That these inhibitors are homologous suggests a common origin. A thorough structural investigation of these inhibitors is proposed. These studies, which include completing the amino acid sequence and pairing the disulfide bonds of the chymotrypsin inhibitor and preparation of crystals of both inhibitors for subsequent x-ray diffraction studies, should provide an interesting comparison of homologous proteins which are directed toward non-homologous target enzymes. Solutions chemical studies will complement structural analysis in providing information about enzyme-inhibitor interactions. A novel approach using an affinity-label derivative of the carboxypeptidase inhibitor to locate the region of carboxypeptidase in contact with inhibitor will be tried. The chymotrypsin-inhibitor interaction will be probed using catalytic cleavage at acid pH to identify the reactive site of the inhibitor. In addition, the strength of binding of the inhibitor to chymotrypsin, several derivatives of chymotrypsin, and trypsin will be determined. The second aspect of this proposal involves using immobilized carboxypeptidase inhibitor to prepare various target enzymes. The method is effective on the bovine, porcine and shrimp carboxypeptidases.$ Other enzymes will be screened for susceptibility to the inhibitor, and those which are affected will be studied to determine whether immobilized inhibitor facilitates their preparation.